Most drug recalls and delays in regulatory approvals result from drug-induced adverse events, and the most frequent reasons for withdrawal of an drug are nephrotoxicity and hepatotoxicity. For example, two widely prescribed non-steroidal anti-inflammatory drugs (NSAIDs), ibuprofen and acetaminophen, are known nephrotoxic1 and hepatotoxic compounds, respectively. However, the mechanisms of NSAID toxicity and the events that produce cell death are poorly understood and our methods of assessing these side effects before use in patients are of limited usefulness.
Current in vitro 2D cell-based assays are easy and inexpensive to perform, as well as amenable to automation and scale up. Unfortunately, 2D systems are often poor predictors of drug-induced hepatotoxicity and can yield false-positives because 2D cultured cells tend to be more sensitive to cytotoxic agents.
Furthermore, the 2D environment does not reflect a natural cellular environment and is not able to model extracellular stresses, such as oxidative and osmotic stress. As an example, kidney cells cultured in 2D in vivo, as compared to those from cell culture models, are likely to be more susceptible to nephrotoxins because of hyperosmolality, but this is difficult (if not impossible) to model in 2D cell cultures. Therefore, 3D tissue models, which can capture cell-cell and cell-matrix interactions and provide a more in vivo-like environment and offer a better platform for toxicity testing.
Materials are being developed that can support three-dimensional (3D) cell culturing conditions. Most of the work in 3D cell culture techniques to date involves rotation of the flasks, the use of an exterior scaffold to which the cells can adhere, the use of magnetic fields to suspend cells, or some combination of these approaches.
For example, Felder in US2005054101, WO2005010162 describes a hydrogel substrate that forms an exterior scaffolding in which cells can grow and be supported in a 3D environment. This introduces an artificial substrate with which cells interact, rather than rapidly promoting cell-cell interactions, and although an improvement over 2D culturing, the scaffolding is likely to perturb the cells and remains in the finished product. Further, cells can grow on or in the microcarriers, but cells cannot be levitated in a manner where all around cell-cell contact and interaction is possible.
There is also a significant level of complexity involved in the fabrication of the microcarriers of Felder, which includes laborious chemistry and the need for complex equipment. Further, algimatrix, one of the main reagents in making the microcarriers, can be a source of endotoxins. Buoyancy control also seems to be relevant to facilitate levitation, and is controlled by the infusion of glass bubbles into the microcarriers, again contributing to complexity and difficulty. Finally, specialized hardware is required for agitation, which is needed achieve gas exchange and to prevent clumping of the microcarriers, and impellers are often used to agitate cells. However, the shear stress resulting from agitation is known to cause cell damage. Furthermore, agitation impairs any magnetic shape control of 3D cultures.
Becker in US2009137018, WO2005003332 uses a coating of bioattractive magnetized core particles, thereby initiating adherence of the biological cells to the magnetized core particles and allowing their suspension in a magnetic field. The coating remains with the cells during culture, thus introducing an unnatural element in the culture and probably perturbing the cells. The inventors contemplate the use of a biodegradable coating that could eventually be eliminated, but none are disclosed, so it is not known if this approach would be successful. Furthermore, because cells are grown on the core of the microcarriers, the levitation of individual cells so they can be brought together by magnetic levitation for the purpose of promoting cell-cell interaction is unlikely to take place. Therefore, it is not obvious that the rapid (hours) assembly of 3D multicellular structures due to cell-cell contact can be demonstrated when using microcarriers. Also, by growing cells on the microcarriers, the co-culture of different cells types, especially by levitating individual cells and then bringing them together magnetically, is not demonstrated. Finally, this system is cumbersome and not suitable for scale-up and high-throughput applications.
A better approach might be to temporarily magnetize cells, allowing for their 3D culture. For example, Akira in US2006063252, WO2004083412, WO2004083416 uses magnetic cationic liposomes (MCL) to magnetize cells by uptake of the liposomes. The magnetized cells are then grown in a sheet on the bottom of a plate using magnetic attraction, and then released for use. However, although able to produce sheets of cells, the cells are still grown on the bottom of a plate, and thus this is not true 3D culturing by magnetic levitation. Further, no functional assay was demonstrated.
Shimizu and Akira et al. recently used magnetic guidance to seed cells onto a decellularized blood vessel. Their study shows encouraging results, but they do not use the magnetized cells as the source of tissue to be decellularized. The magnetized cells are only used to recellularize the decellularized blood vessels.
In patent application WO2010036957 by Souza, cells are levitated in a magnetic field by contacting the cells with a “hydrogel” comprising a bacteriophage with nanoparticles that are responsive to a magnetic field. In particular, filamentous phage, such as fd, fl, or M13 bacteriophage, are used. How the method works is not completely clear, but it is theorized that the phage provide a gel-like structure or assembly that coats the cells, and somehow assists the cells to uptake or adsorb the magnetically responsive nanoparticles. Thus, even when the hydrogel is washed away, the cells remains magnetically responsive, and can be levitated in an appropriate magnetic field. However, although the hydrogel is mostly washed away, the potential for phage infectivity or transfer of genetic material remains, and thus it is desired to provide a material that allows cell uptake or adsorption without the use of phage.
WO2011038370, also by Souza, describes a second generation hydrogel, which completely avoids the use of bacteriophage to enable the magnetization of cells. Furthermore, a variation of the claimed gels is now commercially available at N3D BIOSCIENCES™, under the trade name NANOSHUTTLE.™
This new hydrogel provides a superior method of magnetizing cells without the use of any toxic or infections agents, and the cells remain magnetized when the gel is washed away.
Although some of these approaches are promising, there is still room for improved 3D methods of assessing drug toxicity that more accurately model a natural environment, and that are robust, reproducible and amenable to scale up.
SUMMARY OF THE INVENTIONAbbre-viationDefinitionDfFractal dimension, a statistical quantity that gives an indicationof how completely a fractal appears to fill space and is usedherein to approximate the number of cells in multicellular3D structures generated by MLM, determined by equation 3,below.ECMExtracellular matrixFBSFetal Bovine SerumKga constant called the structure prefactor, which is oftenempirically determined and can vary depending on the systembeing measured and the type of measurement2. Here, itwas arbitrarily chosen as 0.8 since the modeled systemmirrors the experimental data.MLMmagnetic levitation methodMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide],used in a prior art cell viability or proliferation assays based onenzymatic conversion of the substrate MTT to a purple color.HPFhuman primary fibroblastSMCsmooth muscle cellsHEKHuman Embryonic KidneyBrEpicBronchial EpithelialNIRNear-infrared spectroscopy
The present invention provides an improved label-free, real-time cell viability assay. The method uses 3D cell culturing by magnetic levitation and couples the 3D culture technique with image analysis to assess cell viability and cell-cell interaction.
By “culturing” herein, we include culturing single cell types or co-culturing more than one cell type.
Generally speaking, the cell viability detection method is based on the principal that dead cells will lyse, thus either lose any magnetically responsive particles and,therefore, no longer levitate, or the death of cells will cause 3D cell clusters to disintegrate. Thus, only live cells will be imaged in the method. Increasing cell death will result in more, but smaller and more diffuse or porous cell clusters. Furthermore, because fractal dimension is a function of both structure and number of cells N, the replication of viable cells will increase N, consequently increase the fractal dimension of the multicellular structures.
The invention also allows us to monitor and measure cell viability (cell health) or cell-cell interactions in response to any test agent (including cells and extracellular matrix proteins as agents) introduced into culture or an environmental change. The technique is novel in that we combine rapid formation of 3D cell culture by magnetic levitation with image analysis of topological parameters for quantifying cell viability or cell-cell interactions. The method can be simply described, as follows:
1. First the cells are levitated to allow 3D culture growth.
2. Low magnification (4×) photomicrographs are taken of the 3D cells.
3. Those photomicrographs are analyzed using e.g., imageJ software (freeware) to measure e.g., fractal dimension, the number of cell clusters, cell cluster size, tissue opening, and/or total area of cell clusters, and other cell parameters as described throughout. This step can be done at any time during or after the experiment.
4. Next, a drug candidate or an agent (including but not limited to drugs, environmental toxins, growth factors, inhibitors, nanoparticles, homogeneous or heterogeneous mixture of cells, and/or homogeneous or heterogeneous mixture of extracellular matrix proteins and/or peptides) is added to the cells, usually in a dose and/or time dependent configuration (e.g, different samples have differing doses of drug).
5. Steps 2 and 3 are repeated.
6. Finally, the output of the image analysis (e.g., including but not limited to, fractal dimension, number of cell clusters, area size of clusters, total area of clusters) is plotted as a function of time and/or drug concentration and that data is used to determine the effect of the test agent on the cells.
Test agents can be anything whose toxicity is desired to be assayed, including drugs, other cell types, cell components, suspected toxins or any environmental or industrial agent or chemical.
Although described in a linear, stepwise fashion above, this is only for ease of understanding, and usually all samples (zero drug control, plus multiple samples with increasing doses of drug) can be processed in parallel. In the alternative, samples can be grown for 1-7, preferably 2-3 days, to attain a sufficient size, and drug added at a later time point.
Furthermore, although we have used a simple ring magnet herein, it is known how to influence 3D culture shape and structure by varying the magnetic field. Finally, although it is preferred to use magnetic levitation, as described herein, any 3D culturing method can be used in the method.
Preferably, the photo's are taken while the cells are still levitating because this is expected to minimize disruption to the culture. However, this is not essential, and a 3D culture can be photographed when not levitating. This may be particularly appropriate for some assays, such as wound healing assays wherein one measures the rate of closure of a gap or hole in a multicell structure.
Preferably, the control sample image (no drug or agent) is assigned to be a 100% viability reference value, and the lowest dosage that produces total cell death is assigned to be the 0% viability reference value. Thus, any values in between should reflect the toxicity of the drug or agent. These calculations are similar to those used for various colorimetric agents, such as MTT, the difference being that other methods use color and the inventive method uses topological variables, such as fractal dimension, and therefore, avoids the addition of any signal reporting chemical or labeling agent.
As a test of the system, we compared MTT data from 2D cultures with our 3D method (FIG. 3) and found the results to be comparable, although the 3D structure provides a protective effect to cells on the interior of the culture, reflected in a significant increase in cell viability.
In more detail, the invention is a cell viability assay comprising culturing levitated cells to form 3D cell structures, taking photomicrographs of said cells at one or more times (it is not required that cells be levitated during photography, but it is preferred to minimize disruptions to the culture), analyzing said photomicrographs to measure one or more of i) fractal dimension, ii) the number of cell clusters, iii) cell cluster size, iv) cell culture texture, v) total area of cell clusters, and/or vi) rate of wound closure or tissue opening (herein referred as wound closure). Finally, fractal dimension, rate of wound closure, and cell cluster size are directly proportional to cell viability or cell-cell interactions, and the number of cell clusters and total area of cell clusters are inversely proportional to same.
There can also be a plurality of samples of levitated cells, said samples being with and without one or more concentrations of various test agents or having test agents added at one or more times. Generally, a decrease in cell viability with said test agent indicates that the test agent is inhibitory, and wherein an increase in cell viability indicates that said test agent is stimulatory. The same method can assess a large number of variables, including agent toxicity, growth stimulation, mitotic stimulation, wound healing, cell-cell interactions, cell size, cell sprouting, cell structural changes, and the like.
Further, although we have focused on viability and cell-cell interactions herein, the method can also provide important information about cell shape and structure, which correlates with important changes in cell status. For example, some cells become rounder on transformation to a cancerous cell, endothelial cells sprout/elongation during angiongenesis process, and the like.
Further, the method can be combined with other methods, e.g., cell and nuclear staining, and effects on cell size, nuclear/cytomplasmic ratio, cell roundness, and the like can also provide important information about cell status. These assays can either be performed after test agent effects on cell viability and/or cell differentiation (in the case of stem cells) have been ascertained by photomicrography, or separate samples can be prepared for same.
In some embodiments, the invention is a cell viability or cell-cell interaction assay comprising: culturing a 3D cell culture with one or more cell types; taking photomicrographs of said 3D cell culture at one or more times; analyzing said photomicrographs to measure one or more of i) fractal dimension, ii) the number of cell clusters, iii) cell cluster size, iv) cell culture texture, or v) total area of cell clusters, wherein fractal dimension and cell cluster size are directly proportional to cell viability, cell-cell interactions, cell migration, extracellular matrix formation, cell-extracellular matrix interaction, or type and/or number of cells present in the culture, and the number of cell clusters and total area of cell clusters are inversely proportional to cell viability, cell-cell interactions, or cell types present in the culture. Rate of wound closure also correlated directly with cell-cell interactions, cell migration, extracellular matrix formation, cell-extracellular matrix interaction, and viability.
The method can include culturing a plurality of 3D cultures for use as control cultures and a culturing a plurality of 3D cultures for use as test cultures, wherein a test agent is added to said test cultures and assessing the effect of said test agent on said cell viability, cell-cell interactions, cell-extracellular matrix interaction, or cell types present in the culture (when co-culturing cells).
The method can also include adding varying amounts of said test agent to said a plurality of test cultures, taking a plurality of photomicrographs of said 3D cell culture at a plurality of times, and/or washing out said test agent and taking a further plurality of photomicrographs of said 3D cell culture at a further plurality of times.
In preferred embodiments, the cells are levitated with a composition comprising: a) a negatively charged nanoparticle; b) a positively charged nanoparticle; and c) a support molecule, wherein one of said negatively charged nanoparticle or positively charged nanoparticle contains a magnetically responsive material, such as iron or iron oxide, and wherein said support molecule holds said negatively charged nanoparticle and said positively charged nanoparticle in an intimate admixture.
Preferably, the support molecule comprises peptides, polysaccharides, nucleic acids, polymers, poly-lysine, fibronectin, collagen, laminin, BSA, hyaluronan, glycosaminoglycan, anionic, non-sulfated glycosaminoglycan, gelatin, nucleic acid, extracellular matrix protein mixtures, antibody, or mixtures or derivatives thereof, b) wherein said negatively charged nanoparticle is a gold nanoparticle, and c) wherein said positively charged nanoparticle is an iron oxide nanoparticle. Most preferred, the composition is NANOSHUTTLE™.
In some embodiments Df=[log(N)−log(kg)]/log(L/a) and Df is the fractal dimension, kg is the structure prefactor and is a constant, N is the number of cells, a is the diameter of a cell, and L is length of the box. Further,
            Cell      ⁢                          ⁢      viability        =                            D          fsample                -                  D                      f            ⁢                                                  ⁢            100            ⁢            %            ⁢                                                  ⁢            cell            ⁢                                                  ⁢            death                                                D          fcontrol                -                  D                      f            ⁢                                                  ⁢            100            ⁢            %            ⁢                                                  ⁢            cell            ⁢                                                  ⁢            death                                ,and Df sample is the fractal dimension of the test culture, and Dfcontrol is the fractal dimension of the control. However, other methods can be used for calculating same.
In wound closure assays, there are two approaches for generating a wound: one, the 3D culture is perforated to form a wound, and the other 3D culture is mechanically shreaded and then magnetically patterned into a ring structure. In both cases, a test agent can be added to said wounded 3D culture, and
            Wound      ⁢                          ⁢      closure      ⁢                          ⁢              (        %        )              =                  100        ⁢        %            -              (                                            area              ⁢                                                          ⁢              of              ⁢                                                          ⁢                              wound                t                                                    area              ⁢                                                          ⁢              of              ⁢                                                          ⁢                              wound                                  t                  ⁢                                                                          ⁢                  0                                                              ×          100                )              ,wherein area of woundt is the area of the wound at a given time after addition of a test agent, and the area of woundt0 is the area of the wound before said test agent is added.
The cell assay can be used in a variety of applications, including at least to assess the effects of a test agent on toxicity, wound healing, mitotic activity, growth stimulation, cell-cell interactions, viability, or cell structure, and the like.